Germline mutations in TP53 account for approximately 70% of cases of Li-Fraumeni Syndrome (LFS), a familial cancer phenotype associated with breast cancer, sarcomas and other tumors. We hypothesized that LFS kindreds without TP53 mutations might harbor mutations in other components of this DNA damage response pathway, and we have recently shown that a subset of these cases do not have germline mutations in hCHK2, the mammalian homolog of a kinase that regulates the G2 checkpoint in yeast. These observations raise the possibility that other genes in the G2 checkpoint may contribute to genetic may contribute to genetic predisposition to breast cancer. Furthermore, the development of early-onset and bilateral breast cancer in kindreds with kCHK2 mutations suggest that alterations in this gene may occur in women who have evidence of predisposition to breast cancer but who lack the classical manifestations of LFS. We propose three major aims: 1. Mutational analysis of G2 checkpoint genes in a highly selected cohort of patients who have breast cancer in the context of multi-cancer syndromes without germline mutations in p53. The absence of p53 mutations in such families enhances the likelihood of detecting mutations in related genes required for genomic stability. 2. Mutational analysis of hCHK2 and related genes in subsets of the general population at increased risk for breast cancer, but without the extraordinary risk factors demonstrated by LFS kindreds. These patients may have mutations with lower penetranc3e, associated with attenuated cancer phenotypes. 3. Mutational analysis of these genes in breast cancer specimens, to determine their contribution to the development of sporadic breast cancer. Together, these studies will address the contribution of a new class of tumor suppressor genes in both familial and sporadic cases of breast cancer.